1. Technical Field
The present invention relates to the assembly of a microfluidic device for the analysis of biological material, in particular for nucleic acid analysis using PCR-type processes, to which the following treatment will make explicit reference, without this implying any loss in generality.
2. Description of the Related Art
Typical procedures for analyzing biological materials, such as nucleic acid, protein, lipid, carbohydrate, and other biological molecules, involve a variety of operations starting from raw material. These operations may include various degrees of cell separation or purification, cell lysis, amplification or purification, and analysis of the resulting amplification or purification product.
As an example, in DNA-based blood analyses, samples are often purified by filtration, centrifugation or by electrophoresis so as to eliminate all the non-nucleated cells, which are generally not useful for DNA analysis. Then, the remaining white blood cells are broken up or lysed using chemical, thermal or biochemical means in order to free the DNA to be analyzed. Next, the DNA is denatured by thermal, biochemical or chemical processes and amplified by an amplification reaction, such as PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), TMA (transcription-mediated amplification), RCA (rolling circle amplification), and the like. The amplification step allows the operator to avoid purification of the DNA being studied because the amplified product greatly exceeds the starting DNA in the sample.
If RNA is to be analyzed, the procedures are similar, but more emphasis is placed on purification or other means to protect the labile RNA molecule. RNA is usually copied into DNA (cDNA) and then the analysis proceeds as described for DNA.
The amplification product undergoes some type of analysis, usually based on sequence or size or some combination thereof. In an analysis by hybridization, for example, the amplified DNA is passed over a plurality of detectors made up of individual oligonucleotide detector fragments that are anchored, for example, on electrodes. If the amplified DNA strands are complementary to the oligonucleotide detectors or probes, stable bonds will be formed between them (hybridization). The hybridized detectors can be read by observation using a wide variety of means, including optical, electromagnetic, electromechanical or thermal means (the so-called “detection” step).
Other biological molecules are analyzed in a similar way, but typically molecule purification is substituted for amplification, and detection methods vary according to the molecule being detected. For example, a common diagnostic involves the detection of a specific protein by binding to its antibody. Such analysis requires various degrees of cell separation, lysis, purification and product analysis by antibody binding, which itself can be detected in a number of ways. Lipids, carbohydrates, drugs and small molecules from biological fluids are processed in similar ways. However, the following discussion will be focused on nucleic acid analysis, in particular DNA analysis, as an example of a biological molecule that can be analyzed using the devices of the invention.
Integrated microfluidic devices for the analysis of nucleic acids are known, which are based on a die of semiconductor material (the so-called LOC, Lab-On-Chip), integrating a series of elements and structures allowing the variety of functions required for the amplification and identification of oligonucleotide sequences to be carried out.
In detail, as is shown in FIG. 1, a microfluidic device 1 for the analysis of DNA, of the integrated type, comprises a base support 2 (in particular, a PCB—Printed Circuit Board) and a microfluidic die 3. The microfluidic die 3 is carried by the base support 2, which also carries the required electrical connections with the outside.
In greater detail, as shown in FIGS. 2 and 3, the microfluidic die 3 comprises a substrate 4 of semiconductor material and a structural layer 5 arranged on the substrate 4 (for example, a layer of glass coupled to the substrate 4). Inlet reservoirs 6 (numbering four, for example) are defined through the structural layer 5, and are in fluid communication with substrate inlets 7 formed through a surface portion of the substrate 4.
A plurality of microfluidic channels 8 (for example, three for each inlet reservoir 6), buried inside the substrate 4 and each one in communication with a respective substrate inlet 7, connect the substrate inlets 7 with respective substrate outlets 9, also formed through a surface portion of the substrate 4.
A detection chamber 10 is defined in the structural layer 5 at the substrate outlets 9, to which it is fluidically connected. In particular, the detection chamber 10 is adapted to receive a fluid containing pre-processed (for example, via suitable heating cycles) nucleic material in suspension from the microfluidic channels 8, to carry out an optical identification step for nucleic acid sequences. To this end, the detection chamber 10 houses a plurality of so-called “DNA probes” 11, comprising individual filaments of reference DNA containing set nucleotide sequences; more precisely, the DNA probes 11 are arranged in fixed positions to form a matrix (a so-called micro-array) 12 and are, for example, grafted onto the bottom of the detection chamber 10. At the end of a hybridization step, some of the DNA probes, indicated by 11′, which have bound with individual sequences of complementary DNA, contain fluorophores and are therefore detectable with optical techniques (so-called “bio-detection”).
Heating elements 13, for example polysilicon resistors, are formed on the surface of the substrate 4 and extend transversally with respect to the microfluidic channels 8. The heating elements 13 can be electrically connected, in a known manner, to external electrical power sources (here not shown) in order to release thermal power to the microfluidic channels 8, for controlling their internal temperature according to given heating profiles (during the above-mentioned heating cycles). In particular, in FIG. 1, contact pads 14 arranged on the base support 2 at the side of the microfluidic die 3 electrically contact the heating elements 13, which in turn electrically contact electrodes 15 formed on the surface of the base support 2; side covers 16 (“globe-tops”), for example made in resin, cover the contact pads 14 at the sides of the microfluidic die 3.
In use, to avoid contamination of the biological material or its evaporation due to the high temperatures that develop during the heating cycles to which the material is subjected, it is required to seal some or all of the substrate inlets 7, the substrate outlets 9 and the detection chamber 10. For example, during the heating cycles all of the above-mentioned openings must be sealed. Conversely, during operations such as the loading of the biological sample to analyze, at least the substrate inlets 7 must be accessible from the outside. Similarly, the substrate outlets 9 and the detection chamber 10 must be accessible during washing and rinsing operations of the detection chamber 10.
In patent application EP 05112913.8 filed in the name of the same applicant on 23 Dec. 2005, the use of gaskets made of a soft biocompatible material, coupled to elastic clips configured to close with pressure on the lateral edges of the base support 2, is described as releasable seals on regions of the microfluidic device. The elastic clips, for example made of a plastic material, are manually applied by a user in correspondence to regions of interest (in particular, the use of at least two plastic clips is suggested for sealing, one for the substrate inlets 7, and the other for the substrate outlets 9 and the detection chamber 10), and their positioning is facilitated by the presence of specially provided positioning pins on the base support 2. When applied in position, the clips push the gaskets against the openings, to seal them.